Modulation of the urokinase-type plasminogen activator receptor by the beta6 integrin subunit

Over-expression of components of the urokinase system is well documented in cancer and is thought to enable tumour cells to migrate and invade. Changes in integrin expression are also a common feature of tumours and have been linked to changes in protease activity. It has been shown that the alphavbeta6 integrin is neo-expressed in a number of epithelial carcinomas and in wound healing situations. We therefore investigated whether alphavbeta6 is able to modulate a key regulator of proteolysis, the urokinase receptor. We report that epithelial cells expressing full-length alphavbeta6 exhibit decreased urokinase receptor expression and function. Furthermore, this novel modulation requires the C-terminal 11 amino acids of the cytoplasmic tail of the beta6 integrin subunit. Cells expressing alphavbeta3, however, did not affect urokinase receptor expression. De novo expression of beta6 by melanoma cells and beta3 by epithelial cells did not influence urokinase receptor expression or function, suggesting that modulation of urokinase system is both integrin subunit and cell-specific.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells.

Using the polymerase chain reaction, we have isolated cDNA clones that encode a new integrin beta subunit--beta 4. Its cDNA, which is 5676 bp in length, has one long coding sequence (5256 bp), a polyadenylation signal and a poly(A) tail. The deduced sequence of 1752 amino acids is unique among the integrin beta subunits. It contains a putative signal sequence as well as a transmembrane domain that divides the molecule into an extracellular domain at the N-terminal side and a cytoplasmic domain at the C-terminal side. The extracellular domain exhibits a 4-fold repeat of cysteine-rich motif similar to those of other integrin beta subunits. Certain features of the extracellular domain, however, are unique to the beta 4 subunit sequence. Of the 56 conserved cysteine residues found within the extracellular domain of other mature beta subunits, eight such residues are deleted from the beta 4 subunit sequence. The cytoplasmic domain is much larger (approximately 1000 amino acids) than those of other beta subunits (approximately 50 amino acids) and has no significant homology with them. A protein homology search revealed that the beta 4 subunit cytoplasmic domain has four repeating units that are homologous to the type III repetition exhibited by fibronectin. The beta 4 subunit mRNA was expressed primarily in epithelial cells. The restricted expression and the new structural features distinguish the integrin beta 4 subunit from other integrin beta subunits.     

Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the alpha v beta 6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the beta 6 subunit into poorly invasive SCC9 cells to establish the SCC9 beta 6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of beta 6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to alpha v beta 6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-alpha 5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of beta 6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9 beta 6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-alpha v beta 6 antibodies. The expression of beta 6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9 beta 6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-alpha v beta 6 antibodies. These results demonstrate that expression of beta 6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin alpha v beta 6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.     

Integrin αvβ1 Is a Receptor for Foot-and-Mouth Disease Virus

Infection by field strains of Foot-and-mouth disease virus (FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including alphavbeta3 and the epithelial integrin alphavbeta6. In this report we show that the integrin alphavbeta1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by alphavbeta1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric alpha5/alphav integrin subunits, in association with the beta1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of alphavbeta1, not alpha5beta1, thus providing further evidence for the receptor role of alphavbeta1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of alphavbeta1 and alphavbeta6.     

Expression and Role of Integrins in Invasive Activity of Oncotransformed Fibroblasts Differing in Spontaneous Metastasizing

Four closely related lines of RSV-transformed Syrian hamster fibroblasts differing drastically in their spontaneous metastatic capacity were investigated for the surface expression of integrins, in vitro invasion, and production of MMP-2 collagenase. The highly metastasizing HET-SR-2SC-LNM cells differ from the lowly metastasizing parental HET-SR cells in a high level of the surface expression of the collagen-specific alpha1beta1, alpha2beta1, and alphavbeta3 integrins, a high invasive activity, and an increased production of MMP-2. The same properties are characteristic for the actively metastasizing cells of the independent HET-SR-1 line. The lowly metastasizing fibroblasts that are derived from HET-SR-2SC-LNM retain a high level of the expression of the alpha1beta1 and alpha2beta1 integrins, but, unlike the parental line, they exhibit a decreased expression of the alphavbeta3 integrin, invasion in Matrigel, and MMP-2 production. Substrate stimulation of the signal function of the collagen-specific integrins increases the production of MMP-2 by the metastatically active fibroblasts. Inhibition of the signal activity of the integrins by RGD-containing pentapeptide or by genistein reduces markedly in vitro invasion in Matrigel and MMP-2 production. The role of specific properties of the extracellular matrix surrounding tumor cells and of specific surface integrins expressed in these cells in developing of the malignant phenotype is discussed.     

Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.     

Identification of Insulin Receptor Substrate 1 (IRS-1) and IRS-2 as Signaling Intermediates in the α6β4 Integrin-Dependent Activation of Phosphoinositide 3-OH Kinase and Promotion of Invasion

Expression of the alpha6beta4 integrin increases the invasive potential of carcinoma cells by a mechanism that involves activation of phosphoinositide 3-OH kinase (PI3K). In the present study, we investigated the signaling pathway by which the alpha6beta4 integrin activates PI3K. Neither the alpha6 nor the beta4 cytoplasmic domain contains the consensus binding motif for PI3K, pYMXM, indicating that additional proteins are likely to be involved in the activation of this lipid kinase by the alpha6beta4 integrin. We identified insulin receptor substrate 1 (IRS-1) and IRS-2 as signaling intermediates in the activation of PI3K by the alpha6beta4 integrin. IRS-1 and IRS-2 are cytoplasmic adapter proteins that do not contain intrinsic kinase activity but rather function by recruiting proteins to surface receptors, where they organize signaling complexes. Ligation of the alpha6beta4 receptor promotes tyrosine phosphorylation of IRS-1 and IRS-2 and increases their association with PI3K, as determined by coimmunoprecipitation. Moreover, we identified a tyrosine residue in the cytoplasmic domain of the beta4 subunit, Y1494, that is required for alpha6beta4-dependent phosphorylation of IRS-2 and activation of PI3K in response to receptor ligation. Most importantly, Y1494 is essential for the ability of the alpha6beta4 integrin to promote carcinoma invasion. Taken together, these results imply a key role for the IRS proteins in the alpha6beta4-dependent promotion of carcinoma invasion.     

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.     

A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function.

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha5beta1-mediated phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal for integrin crosstalk.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

Background

Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results

In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion

A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.     

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Engineered fibronectin type III domain with a RGDWXE sequence binds with enhanced affinity and specificity to human alphavbeta3 integrin

Fibronectin is an extracellular matrix protein with broad binding specificity to cell surface receptors, integrins. The tenth fibronectin type III domain (FNfn10) is a small, autonomous domain of fibronectin containing the RGE sequence that is directly involved in integrin binding. However, in isolation FNfn10 only weakly bind to integrins. We reasoned that high-affinity and high-specificity variants of FNfn10 to a particular integrin could be engineered by optimizing residues surrounding the integrin-binding RGD sequence in the flexible FG loop. Affinity maturation of FNfn10 to alphavbeta3 integrin, an integrin up-regulated in angiogenic endothelial cells and in some metastatic tumor cells, yielded alphavbeta3-binding FNfn10 mutants with a novel RGDWXE consensus sequence. We characterized one of the RGDWXE-modified clones, FNfn10-3JCLI4, as purified protein. FNfn10-3JCLI4 binds with high affinity and specificity to purified alphavbeta3 integrin. Alanine scanning mutagenesis suggested that both the tryptophan and glutamic acid residues following the RGD sequence are required for maximal affinity and specificity for alphavbeta3. FNfn10-3JCLI4 specifically stained alphavbeta3-positive cells as detected with flow cytometry and it inhibited alphavbeta3-dependent cell adhesion. As with the anti-alphavbeta3 antibody LM609, FNfn10-3JCLI4 can interfere with in vitro capillary formation. Taken together, these data show that FNfn10-3JCL14 is a specific, high-affinity alphavbeta3-binding protein that can inhibit alphavbeta3-dependent cellular processes similar to an anti-alphavbeta3 monoclonal antibody. These properties, combined with the small, monomeric, cysteine-free and highly stable structure of FNfn10-3JCLI4, may make this protein useful in future applications involving detection and targeting of alphavbeta3-positive cells.     

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.     

ADAM15 decreases integrin alphavbeta3/vitronectin-mediated ovarian cancer cell adhesion and motility in an RGD-dependent fashion

We have recently described that integrin alphavbeta3 upon interaction with its major extracellular matrix ligand vitronectin induces adhesion, motility, and proliferation of human ovarian cancer cells. Due to the important function of alphavbeta3 in cancer cell biology, it has been the effort of many scientific approaches to specifically target alphavbeta3-mediated cell adhesion and tumorbiological effects arising thereof by synthetic integrin antagonists. More recently, proteins of the ADAM family have been recognized as naturally occurring integrin ligands. Among those, human ADAM15 which encompasses the integrin binding RGD motif was shown to interact with integrin alphavbeta3. Thus, we investigated in human ovarian OV-MZ-6 cancer cells, expressing both ADAM15 and alphavbeta3, whether ADAM15 might affect alphavbeta3-mediated tumorbiological effects. We stably (over)expressed ADAM15 or its extracellular domain in OV-MZ-6 cells as well as respective ADAM15 mutants containing the tripeptide SGA instead of RGD. Cells (over)expressing ADAM15-RGD exhibited a significantly reduced alphavbeta3-mediated adhesion to vitronectin. Also, a significant time-dependent decline in numbers of cells cultivated on vitronectin was noticed. This effect was found to be rather due to impaired alphavbeta3-mediated cell adhesion than decreased cell proliferation rates, since de novo DNA synthesis was not significantly altered by elevated ADAM15 expression. Moreover, a substantially decreased random cellular motility was noticed as a function of ADAM15 encompassing an intact RGD motif. In conclusion, our results point to a physiological role of ADAM15 as a natural binding partner of integrin alphavbeta3 thereby loosening tumor cell adhesion to the underlying matrix and regulating tumor cell migration and invasion.